HIV-protease inhibitors suppress skeletal muscle fatty acid oxidation by reducing CD36 and CPT1 fatty acid transporters

Abstract

Infection with human immunodeficiency virus (HIV) and treatment with HIV-protease inhibitor (PI)-based highly active antiretroviral therapies (HAART) is associated with dysregulated fatty acid and lipid metabolism. Enhanced lipolysis, increased circulating fatty acid levels, and hepatic and intramuscular lipid accumulation appear to contribute to insulin resistance in HIV-infected people treated with PI-based HAART. However, it is unclear whether currently prescribed HIV-PIs directly alter skeletal muscle fatty acid transport, oxidation, and storage. We find that ritonavir (r, 5micromol/l) plus 20micromol/l of atazanavir (ATV), lopinavir (LPV), or darunavir (DRV) reduce palmitate oxidation(16-21%) in differentiated C2C12 myotubes. Palmitate oxidation was increased following exposure to high fatty acid media but this effect was blunted when myotubes were pre-exposed to the HIV-PIs. However, LPV/r and DRV/r, but not ATV/r suppressed palmitate uptake into myotubes. We found no effect of the HIV-PIs on FATP1, FATP4, or FABPpm but both CD36/FAT and carnitine palmitoyltransferase 1 (CPT1) were reduced by all three regimens though ATV/r caused only a small decrease in CPT1, relative to LPV/r or DRV/r. In contrast, sterol regulatory element binding protein-1 was increased by all 3 HIV-PIs. These findings suggest that HIV-PIs suppress fatty acid oxidation in murine skeletal muscle cells and that this may be related to decreases in cytosolic- and mitochondrial-associated fatty acid transporters. HIV-PIs may also directly impair fatty acid handling and partitioning in skeletal muscle, and this may contribute to the cluster of metabolic complications that occur in people living with HIV.

Fig. 1. Concentration-dependent effects of HIV-PIs on ¹⁴C]-palmitate oxidation in skeletal muscle cells

L6 and C2C12 cells were exposed to HIV-PIs in the absence and presence of RTV (5 μM) and [¹⁴CO₂] production (nmol/mg/h) measured. A. IDV. B. LPV/RTV. C. ATV/RTV. Results are mean ± SEM (n = 3 in each group). *PI groups significantly different from Con (DMSO) group (p ≤ 0.05).

Fig. 2. HIV-protease inhibitor treatment reduces [¹⁴C]-palmitate oxidation in C2C12 myotubes

C2C12 myotubes were treated for 18 h with HIV-PIs in combination (ATV/r, LPV/r, and DRV/r; 20/5 μmol/l, respectively) and [¹⁴CO₂] production measured. Results are mean ± SEM (n = 3 in each group). *PI groups significantly different from Con (DMSO) group (p ≤ 0.05).

Fig. 3. Lipid accumulation in C2C12 myotubes following a fatty acid load

Oil red-O staining of neutral lipids in C2C12 myotubes treated with a fatty acid load (50/25 μmol/l, 100/50 μmol/l, and 200/100 μmol/l; oleate/palmitate, respectively) in a serum-free DFM for 5, 18, or 48 h.

Fig. 4. Fatty acid load increases [¹⁴C]-palmitate oxidation in C2C12 myotubes

C2C12 myotubes were treated for 18 h with 200/100 μmol/l oleate /palmitate and [¹⁴CO₂] production measured. Results are mean ± SEM (n = 3 in each group). *Oleate/palmitate group significantly different from Con group (p ≤ 0.05).

Fig. 5. HIV-protease inhibitor treatment followed by fatty acid load decreases [¹⁴C]-palmitate oxidation in C2C12 myotubes

C2C12 myotubes were treated for 18h with PI’s in combination (ATV/r, LPV/r, and DRV/r; 20/5 μmol/l, respectively) followed by a fatty acid load (200/100 μmol/l oleate/palmitate) for 18 h and [¹⁴CO₂] production measured. Results are mean ± SEM (n = 3 in each group). *PI groups significantly different from Con (DMSO) group (p ≤ 0.05).

Fig. 6. Lipid accumulation in C2C12 myotubes treated with HIV-PIs

C2C12 myotubes were treated for 18 h in the presence of vehicle (DMSO) alone (Con) or with HIV-PIs in combination (ATV/r, LPV/r, and DRV/r; 20/5 μmol/l, respectively). The cells were then washed with HBSS and the Con cells were incubated with vehicle alone and the HIV-PI pretreated cells were incubated with 200/100 μmol/l oleate/palmitate for an additional 18 h. In some experiments, the myotubes were treated with only the fatty acids (FA) for 18 h (FA18) or 36 h (FA36). At the end of the incubation period, lipid in the cells was stained with 3% Oil Red O and quantitated as described in Methods section. The presented data are mean ± SEM of %Oil Red O stain/μg protein (n =3). (*^.#FA groups significantly different from Con group, p < 0.0001 and p < 0.05, respectively).

Fig. 7. HIV-protease inhibitor treatment decreases [¹⁴C]-palmitate uptake in C2C12 myotubes

C2C12 myotubes were treated for 18 h with HIV-PIs in combination (ATV/r, LPV/r, and DRV/r; 20/5 μmol/l, respectively) followed by a fatty acid load (200/100 μmol/l oleate/palmitate) for 3 h. Myotubes were harvested and assayed for [¹⁴C]-palmitate incorporation. Results are mean ± SEM (n = 3 in each group). *PI groups significantly different from Con (DMSO) group (p ≤ 0.05).

Fig. 8. Effects of HIV-protease inhibitor treatment on proteins involved in fatty acid transport and metabolism in C2C12 myotubes

Representative immunoblots for FABPpm, FATP1, and FATP4 (A), CD36 (B), CPT1 (C), and SREBP-1 (D) in whole cell lysates of C2C12 myotubes following treatment for 18 h with vehicle (DMSO) alone (Con) or with PI’s in combination (ATV/r, LPV/r, and DRV/r; 20/5 μmol/l, respectively). Tubulin (A) or GAPDH (B-D) were used as loading controls. E. Quantitation of immunoreactive CD36, CPT1, and SREBP-1 bands. The target immunoreactive bands were normalized to GAPDH loading control and the data are presented as fold-change relative to untreated Con sample. (*Significantly different from corresponding Con group, p < 0.0001, n =3).